Synthetic oligonucleotides play a key role in molecular biology research, useful especially for DNA sequencing, DNA amplification, and hybridization. A novel "one pot" enzymatic method is described to replace both the obsolete enzymatic methods and the current phosphoramidite chemical method. This new method promises increased throughput and reliability, ease of automation, and lower cost.
Before the introduction of the phosphoramidite chemical method in 1983, enzymatic methods were used for the synthesis of oligonucleotides. Historically, two distinct enzymatic approaches have been employed as summarized in FIG. 1. These enzymatic methods have been abandoned, however, in favor of the superior phosphoramidite chemical method.
The first enzymatic approach is the "uncontrolled" method. As depicted in FIG. 1A, a short oligonucleotide primer is incubated with the desired nucleotide and a nucleotidyl transferase. At the end of the optimal incubation period, a mixture of oligonucleotide products containing different numbers of bases added to the primer (i.e. primer, primer +1,primer +2 . . . ) is obtained. The desired product, the primer with one added base, is purified using either electrophoresis or chromatography. The process of enzyme incubation and oligonucleotide purification is repeated until the desired oligonucleotide is synthesized. Examples of the use of this approach are: (1) Polynucleotide Phosphorylase ("PNP") and ADP, GDP, CDP, and UDP have been used to make oligodeoxyribonucleotides in accordance with the following reaction: EQU primer+rNDP.fwdarw.(primer-rN)-3'-OH+PO.sub.4
Shum et al, Nucleic Acids Res., 5(7): 2297-311 (1978), and (2) Terminal deoxynucleotidyl Transferase and the nucleotides dATP, dGTP, dCTP, and dTTP have been used to make oligodeoxyribonucleotides in accordance with the following reaction: EQU primer+dNTP.fwdarw.(primer-dN)-3'-OH+pyrophosphate
Schott et al, Eur. J. Biochem, 143: 613-20 (1984). The flaws of the "uncontrolled" approach are the requirement for cumbersome manual purification of the primer+1 product after each coupling cycle, poor yields of the desired primer+1 product, and inability to automate.
The second enzymatic approach is the "blocked" method, shown in FIG. 1B. The nucleotide used in the extension step is blocked in some manner to prevent the nucleotidyl transferase from adding additional nucleotides to the oligonucleotide primer. After the extension step, the oligonucleotide product is separated from the enzyme and nucleotide, and the blocking group is removed by altering the chemical conditions or by the use of a second enzyme. The oligonucleotide product is now ready for the next extension reaction. Examples of this approach are: (1) PNP and NDP-2'-acetal blocked nucleotides have been used to make oligoribonucleotides. The acetal blocking group is removed under acidic conditions (Gilham et al, Nature, 233: 551-3 (1971) and U.S. Pat. No. 3,850,749), (2) RNA ligase and the blocked nucleotide App(d)Np (or ATP+3',5'-(d)NDP) have been used to make oligoribonucleotides and oligodeoxyribonucleotides. The 3'-phosphate blocking group is removed enzymatically with a phosphatase such as alkaline phosphatase (T. E. England et al, Biochemistry, (1978), 17(11), 2069-81; D. M. Hinton et al, Nucleic Acids Research, (1982), 10(6), 1877-94).
The advantage of the "blocked" method over the "uncontrolled" method is that only one nucleotide can be added to the primer. Unfortunately, the "blocked" method has several flaws which led to its abandonment in favor of the chemical method. The "blocked method", like the "uncontrolled" method, requires the purification of the oligonucleotide product from the reaction components after each coupling cycle.
In the first approach, using PNP, the oligonucleotide is exposed to acid to remove the acid-labile acetal blocking group. Oligonucleotide product must be purified and redissolved in fresh buffer in preparation for the next polymerization reaction for two reasons: (1) PNP requires near neutral pH conditions whereas acetal removal requires approximately pH 1; and (2) the product of the polymerization reaction, PO.sub.4, must be removed or it will cause phosphorolysis of the oligoribonucleotide catalyzed by PNP.
In the second approach, using RNA ligase, the art teaches that oligonucleotide product needs to be purified after each cycle because the dinucleotide App(d)N, formed by phosphatase treatment of App(d)Np, is still a suitable substrate for RNA ligase and must be completely removed prior to addition of RNA ligase in the next cycle. England et al., Proc. Natl. Acad. Sci. USA, 74(11): 4839-42 (1977). Hinton et al. emphasize the importance of purifying oligonucleotide product after each cycle by stating: "This elution profile [a DEAE-sephadex chromatogram of oligodeoxyribonucleotide product] also demonstrates the absence of either significant contaminating products arising from nucleases or of the reaction intermediate, A-5'pp5'-dUp. The absence of such substances is critical if this general methodology is to be useful for synthesis." Hinton et al, Nucleic Acids Research, 10(6):1877-94 (1982). The art also teaches that nucleoside and phosphate by-products generated by phosphatase incubation of the RNA Ligase reaction mixture substantially inhibit RNA Ligase activity and must be removed prior to subsequent RNA ligation steps in order to work usefully. Middleton et al., Anal. Biochem., 144:110-117 (1985).
Two modifications have been devised for the "blocked" method to improve the oligonucleotide product yield and to speed required oligonucleotide product purification after each coupling cycle. The first modification was .the use of a branched synthetic approach (Oligonucleotide Synthesis: a practical approach, M. J. Gait editor, (1985), pp. 185-97, IRL Press). This approach improved the yield of final oligonucleotide product, but intermediate purification of oligonucleotide after each coupling cycle was still required. The second modification was the covalent attachment of the primer chain to a solid phase support (A. V. Mudrakovskaia et al, Bioorg. Khim, (1991), 17(6), 819-22). This allows the oligonucleotide to be purified from all reaction components simply by washing the solid phase support column. However, product yields are still low, and primer chains which do not couple during a cycle are not removed and are carried over to the next coupling cycle. It appears that the poor coupling efficiency results from steric problems encountered by the enzyme in gaining access to the covalently bound primer chain. Unfortunately, it is not possible to combine these two modifications in an automated manner. The current phosphoramidite chemical method for oligonucleotide synthesis also utilizes a solid phase support to facilitate oligonucleotide purification after each coupling reaction.
The present invention provides a method for enzymatic oligonucleotide synthesis which is preferably performed entirely in a single tube, requiring only temperature control and liquid additions, and not requiring intermediate purifications or solid phase supports. This method is well suited for automation on a liquid handling robot apparatus, allowing the simultaneous preparation of a thousand oligonucleotides per day in microtiter plates. This capability dwarfs the best commercially available instrument which can prepare only four oligonucleotides simultaneously with the phosphoramidite method (Applied Biosystems, Inc.).